VALIDATION OF THE USE OF SUS SCROFA (DOMESTIC ......3.4 STR profile of the bone sample that was...
Transcript of VALIDATION OF THE USE OF SUS SCROFA (DOMESTIC ......3.4 STR profile of the bone sample that was...
VALIDATION OF THE USE OF SUS SCROFA (DOMESTIC PIG) DNA AS A
RESEARCH TOOL IN A FORENSIC LABORATORY
By:
Nathan Bennoit
Submitted in partial fulfillment of the course FORS 4095 E
Department of Forensic Science Laurentian University
Sudbury, Ontario P3E 2C6
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Nathan R. Bennoit, B.Sc. (Hons.), and Michele Bobyn, B.Sc., M.Sc. VALIDATION OF THE USE OF SUS SCROFA (PIG) DNA AS A RESEARCH TOOL IN A FORENSIC LABORATORY
ABSTRACT: For a number of years, pigs have been used in a variety of forensic disciplines as a
research tool, such as in forensic anthropology. However, pigs have not been used as a research
tool in forensic DNA analysis. The use of pig DNA is becoming common in different areas of
forensic science such as wildlife management but the transition to forensic DNA research has
not happened yet. This paper proposes that pig DNA can be used in a forensic setting for
research purposes in a lab that also conducts case work. The methods followed for the
development of an STR profile using a commercial multiplex kit for pigs are the same as the
standard procedure in a forensic Human DNA laboratory. The validation showed that pig DNA
can be analyzed with the equipment currently used for human samples and it does not cross
contaminate human samples. These factors would help small labs conduct both research and
casework within the same laboratory.
KEYWORDS: Forensic science, Forensic Biology, DNA analysis, Validation study, Sus Scrofa, STR, Animal type Pig PCR amplification kit
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ACKNOWLEDGEMENTS
Thank you to Professor Bobyn for the time and equipment that was lent to this study
and towards the completion of the research; additionally, for all of the support and guidance
that was provided throughout the study. Thank you to Dr. Merritt for allowing me to use his
laboratory space and equipment to complete my research. Thank you to the Canadian Food
Inspection Agency for providing the pigs’ blood for this experiment. Thank you to Alison at
Rowan-tree Farm for donating pork samples. Thank you to the Tarini Brothers for donating pork
bones.
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TABLE OF CONTENTS
Abstract .................................................................................................................................i
Acknowledgements ................................................................................................................ii
Table of Contents ...................................................................................................................iii
List of Tables ..........................................................................................................................iv
List of Figures .........................................................................................................................v
Glossary ..................................................................................................................................vi
Chapter 1: INTRODUCTION AND BACKGROUND
1.1 Introduction and statement of problem ........................................................................1
1.2 General Background .......................................................................................................1
1.3 Goal of the study ............................................................................................................5
Chapter 2: MATERIALS AND METHODS
2.1 Sample Collection ...........................................................................................................7
2.2 DNA Extraction ...............................................................................................................8
2.3 Quantification of DNA ....................................................................................................12
2.4 PCR Amplification and Electrophoresis ..........................................................................17
2.5 Statistical Tests Used ......................................................................................................19
Chapter 3: RESULTS
3.1 Results ............................................................................................................................21
Chapter 4: DISCUSSION
4.1 Points of Discussion........................................................................................................42
4.2 Limitations of the Study .................................................................................................47
Chapter 5: CONCLUSIONS
5.1 Summation .....................................................................................................................49
5.2 Recommendations .........................................................................................................49
References .............................................................................................................................51
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LIST OF TABLES
TABLE
2.1 Complete list of all extracted DNA samples ..............................................................14
2.2 The different reagents and volumes for one sample to be injected in the
Applied Biosystems real time PCR instrument ..........................................................15
2.3 Dilutions that were made to each of the extracted samples to make
the approximate concentration of DNA 2.5ng per µl ................................................16
3.1 Concentration of DNA in each of the Pig muscle and bone samples ........................23
3.2 Concentration of the extracted DNA from the pig blood samples ............................24
3.3 Average heterozygous and homozygous Peak height for each dilution ..................35
3.4 Peak height to stutter ratio of the different loci in the STR profiles .........................41
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LIST OF FIGURES
FIGURE
3.1 STR profile of the positive control that came in the Animaltype pig kit ....................25
3.2 Negative amplification blank STR profile ...................................................................26
3.3 STR profile for the meat sample that was obtained from the pork bone ..................27
3.4 STR profile of the bone sample that was taken from the pork bone .........................28
3.5 STR profile of pig blood sample 1 with no dilution ....................................................29
3.6 STR profile of pig blood sample 1 with a 1:10 dilution ..............................................30
3.7 STR profile of pig blood sample 1 with a 1:25 dilution ..............................................31
3.8 STR profile of pig blood sample 1 with a 1:50 dilution ..............................................32
3.9 STR profile of pig Blood sample 1 with a 1:100 dilution ............................................33
3.10 STR profile of pig blood sample 1 with a 1:200 dilution ............................................34
3.11 50%/50% mixture of DNA between pig blood sample 3 and bone sample 1 ............37
3.12 25%/75% mixture of DNA between pig blood sample 3 and bone sample 1 ............38
3.13 10%/90% mixture of DNA between pig blood sample 3 and bone sample 1 ...........39
3.14 5%/95% mixture of DNA between pig blood sample 3 and bone sample 1 .............40
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Glossary
Allele One of two or more forms of a gene that are found in the same region of the
chromosome on homologous chromosomes
Locus The specific location on a chromosome where a gene or allele is found
PCR Stands for Polymerase Chain Reaction. It is the process of heating and cooling a
double stranded piece of DNA to have it denature and anneal in the presence of
Taq polymerase and nucleotides to replicate specific regions of the DNA strand.
Primer A short strand of DNA that is capable of binding to a specific region of single
stranded DNA known as the complementary sequence.
RFU Stands for Relative fluorescence units. RFUs are the units of measurement for
peak heights in a DNA profile to show the relative amount of a certain piece of
DNA.
STR Stands for Short Tandem Repeat. A short tandem repeat is an area on a
chromosome where a nucleotide sequence 2-13 base pairs long is repeated
many times in tandem. These STR regions of the genome are the areas that are
targeted for PCR amplification and are the basis of DNA fingerprinting.
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CHAPTER 1
INTRODUCTION AND BACKGROUND
1.1 Introduction and Statement of Problem
The use of pigs in forensic research is not a novel idea. The use of pigs can be widely
seen in forensic entomology, anthropology, toxicology and other areas [1-3]. In these
disciplines, pigs are used instead of human bodies for research on different aspects of
decomposition. This type of research is done with pigs because human bodies are often in short
supply or difficult to acquire. Despite this fact, the use of pigs in forensic DNA research has been
limited. Using pigs in forensic DNA research would be vital in aiding the research of the
degradation of DNA on a whole corpse where human bodies are not a practical option.
Additionally, pig samples cost far less than human samples.
1.2 General Background
Pigs have been used in forensic DNA analysis for wildlife management and animal
protection. For example, Robino et al [4] outlined the process of identifying pig samples in a
veterinary malpractice case. Another study looked at the same STR kit (Animal-type Pig kit) for
wildlife protection and conservation [5]. These articles demonstrate that animal DNA analysis
methods have become common practise in certain forensic fields. (e.g., border security meat
analysis and the food industry [6].) The possible applications of Pig DNA for forensic DNA
research have not been demonstrated. The techniques and procedures to analyze animal DNA
are becoming cheaper and quicker, reaching the same level of human DNA analysis is [7]. In
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order for the use of pig DNA to become relevant in an accredited DNA laboratory setting, the
pig DNA must be able to be analyzed using standard equipment that would be found in a
forensic DNA laboratory.
In a standard DNA laboratory there is a typical flow of the samples through the lab. It
starts with the extraction of the DNA from the. This step is not species-specific as most nuclear
DNA can be extracted using the same method. Once the DNA is extracted the DNA has to be
quantified, allowing the analyst to know how much DNA is present. The quantitation step uses
a real time PCR instrument and has species-specific primers that look for one or more specific
sequences of nucleotides and bind to those sites. The real time PCR instrument will then
monitor the quantity of the selected target sequence in real time. The next step is PCR and
amplification of specific regions of the genome that will be analyzed with electrophoresis. This
step is also species-specific as different primers will have to be used to target different loci for
different species. Lastly, the amplified DNA is run through a capillary electrophoresis device to
get the base pair size of each of the sequences of DNA. A computer software program then
designates each of the sequences of DNA an allele value based on the size and the fluorescent
tag associated with the sequence.
Because the analyst has to use the same equipment that is used on human DNA, it must
be shown that there is no cross contamination from pig DNA to human DNA. That is to say that
the instruments won’t detect pig DNA as human DNA and come up with a false positive. This
means that there needs to be specific DNA markers that search for and bind to single stranded
DNA that has the complementary sequence. The term complementary strand means the
sequence is optimal for binding to it, Adenine binds with Thymine and Cytosine binds with
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Guanine. (e.g., ATTGC will have the complementary sequence TAACG.) However the marker will
need to be found in all Pig DNA sequences [8] and only pig DNA. Novel STR markers have been
identified in pigs that would allow them to be classified individually [8]. An STR marker is a
sequence of DNA that flanks a region of DNA that has a short sequence (usually a 4-nucleotide
sequence) that is repeated multiple times consecutively and that repeats a different number of
times in different individuals. One such commercial multiplex kit is the Biotype Diagnostic
Gmbh: Animaltype Pig PCR Amplification kit for STR amplification [14]. This kit is capable of
being used with the current equipment that is found in the Laurentian University Forensic DNA
Lab.
A second reason that pig DNA may not be commonly used in a forensic laboratory is
because the reason for using pig DNA is not recognized. With new major areas of study in
forensic DNA analysis focusing on touch DNA [9] there is no shortage of touch DNA that can be
easily accessed from humans. Yet, the use of pigs will be more relevant when a decomposed or
very complex or compromised sample is found at a crime scene. Pigs will be useful in these
types of cases because using pigs will permit ethical clearance for the research to commence.
Pigs are killed every day in the food industry and research but the number of humans that die
and donate their bodies to science are far more limited. Decomposition research using pigs
would allow researchers to identify areas of the genome that degrade the slowest, and a
proper procedure may be developed that would be done with human DNA that has been
exposed to the same degradation factors.
An additional positive to doing research with pig DNA instead of human is that pig DNA
cannot contaminate a human sample because the STR markers will not match and are human
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specific [10]. Some of the human STR markers that appear to not have any cross contamination
from other species are: CSF1PO, TPOX, THO1, HPRTB, FESFPS, vWF and F13A01 [10]. Primate
species (Gorilla and Chimpanzee) appear to have STR amplification peaks that fall outside of the
bins (Expected base pair sizes to find alleles) of humans [10]. This means that a lab that is
primarily research based, but that also does pay as you go service for police agencies, does not
need to worry that traces of their research will contaminate the evidence that they are
examining.
Because the use of pig DNA in a forensic laboratory is a novel technique, it will need to
be validated. The Scientific Working Group on DNA Analysis Methods (SWGDAM) has extensive
guidelines for validating any novel DNA analysis methods [11]. The guidelines outline that the
method must be species specific, sensitivity studies must be done, precision and accuracy must
be shown and it must work with case type samples [11]. These validations are of the utmost
importance to demonstrate that pigs are a reliable model in forensic DNA analysis. They are
important because any lab must prove that their methods are reliable and reproducible so that
they can be tried and tested in court [12]. These high standards are the reason forensic DNA
analysis is considered to be the gold standard for forensic laboratories. This is why it is
important that the pig DNA analysis can use the exact same, or very similar, procedures as
human DNA. If pig DNA research is held to the same standard as human DNA then the model
will be extremely reliable.
The implication of this research is that it has the potential to open up opportunities for
research for laboratories that do not have access to sufficient funds or access to human
samples for research purposes. Research using pig samples will have fewer ethical issues to deal
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with than research with human samples thus; pig samples would be more accessible. Whole pig
bodies can be bought from a butcher or a farm and then used in research. Some research could
include decomposition timelines for the quality and quantity of DNA that can be extracted from
hard or soft tissue. The use of pigs could also allow for research into what tissue samples yield
the most DNA and best quality after decomposing for long durations. This could create a guide
line for pathologists to know which samples they should collect first to ensure that a profile can
be obtained from bodies that are found at crime scenes.
1.3 Goal of the Study
The aim of this study is to develop a procedure that will allow pig samples to be
analyzed in a forensic setting identical to the way that human samples are currently. The goal is
that only some of the materials would change (STR marker kits) and no other part of the
procedure would be affected. To achieve this goal, it must be demonstrated that pig DNA can
be extracted and that we can generate an STR profile for individual pigs analogous to STR
profiles developed for forensic samples of human origin. From there, samples from different
tissue types will have to be tested repeatedly to show precision. Different samples from
different pigs will also need to be used to assure that the STR kits have alleles that differ from
pig to pig yielding different DNA profiles. Discrimination is essential for forensic DNA samples
for mixture analysis and also for identification purposes of individuals. Lastly, the validation will
have to include samples that are of tissues found at crime scenes such as blood or saliva and
the samples will have to be on different substrates and possibly mixed with other samples to
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prove that the techniques work with forensic case type samples.
Due to limited resources a small sample size, less than 40 were used. As a result, test
statistics were also limited. A comparison of the mean peak heights, and the amount of
background noise of the STR profiles, were undertaken. The stutter ratio was also calculated.
Stutter is a peak that is 4 base pairs shorter than the actual peak which is created from slippage
during PCR.
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CHAPTER 2
MATERIALS AND METHODS
2.1 Sample Collection
The samples that were analyzed in this study were acquired from two different sources.
The meat and bone samples were from a pork bone that was acquired from a local butcher
(Tarini Brothers, Sudbury Ontario). Blood samples were taken from five different pigs that were
purchased from Canadian food inspection agency (Canadian Food Inspection Agency, Ottawa,
Ontario). The pigs were bled as part of a routine collection of blood by the Canadian Food
Inspection Agency in Ottawa. During this routine collection procedure, the pigs were not
euthanized. The blood samples were stored and shipped in lavender topped test tubes. Each
tube was treated with Ethylenediaminetetraacetic acid (EDTA), a blood preservative and
anticoagulant. The five test tubes of pig blood were stored in a refrigerator at 4oC until needed
for DNA extraction.
The pork bone that was used for the collection of the meat and bone samples was
uncooked and acquired from the Tarini Brothers in Sudbury. To prepare the meat sample the
pork bone was thawed as it was previously frozen at -18oC for storage purposes. Once the bone
had been thawed the meat on the bone was teased off of the bone using a new sterile scalpel.
The meat was divided into 10 approximately equal portions. Each of the portions of the meat
was placed into a sterile 1.5 ml microfuge tube and labelled 1 through 10. These samples were
then frozen at -18oC until needed for DNA extraction.
The third type of tissue sample, bone was collected off of the same pork bone that the
meat samples were obtained from. A hammer was used to strike the bone and dislodge a small
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portion. A spherical piece of bone approximately two centimeters in diameter came off the
pork bone. The small piece of bone was ground in a black and decker coffee bean grinder (Black
and Decker, Towson, Marland). The grinder was cleaned with a water and bleach solution
before and after each use. The resulting flakes of bone were collected and divided up into five
sub-samples. Each sub-sample was placed into its own 1.5 ml microfuge tube and marked as
bone sample 1 through 5 and frozen at -18oC for storage.
2.2 DNA Extraction
The entire set of DNA extractions were carried out with AutoMate ExpressTM Forensic
DNA Extraction system instrument from Thermo Fisher Scientific (Waltham, Massachusetts,
United States). The extraction using the meat samples was done with two different magnetic
bead DNA extraction procedures: the PrepFiler ExpressTM Forensic DNA Extraction Kit and the
PrepFiler Express BTATM Forensic DNA Extraction Kit (Applied BiosystemsTM). The first was
called the ‘bodily fluid extraction’ and the second was called the ‘Bone, Tooth and Adhesive
(BTA) extraction’. Both of these procedures were preprogrammed into the AutoMate Express
instrument. Both of the extraction methods were used because the meat sample seemed to fall
within the recommended sample types for each of the extraction techniques. It was unclear
which extraction technique would be most beneficial in extracting the DNA from the meat
sample. It was expected that the BTA extraction technique would yield more DNA because the
process was more vigorous and gain access to more of the nuclear DNA from the muscle tissue.
The procedure of the bodily fluid extraction technique involved some sample
preparation beforehand. In order to prepare the samples for extraction of DNA the samples
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were placed into a tube called the Lysep elution tube. This elution tube had a hole in the
bottom with a filter and a tube connected to the bottom of the Lysep elution tube. The
combination of the Lysep elution tube and bottom tube formed what was termed the Lysep
column. Approximately 0.6g of the meat sample was placed into each of the Lysep columns.
Each of the columns had the addition of 500µl of the PrepFiler lysis solution that was made with
500µl of the provided PrepFiler lysis buffer and 5µl of 1M Dithiothreitol (DTT). Once each of the
Lysep columns had the necessary solutions, the samples were placed into the Thermomixer
from Eppendorf (Eppendorf, Hamburg, Germany). The Thermomixer was set to 70oC and
750rpm. The five meat samples that were undergoing the bodily fluid extraction were placed
into the thermomixer for 40 minutes. At the end of the 40 minutes the samples were taken out
of the Thermomixer and were placed into a centrifuge where they were spun at 10,000x the
force of gravity for two minutes. After the two minutes of centrifugation the samples were
removed from the centrifuge and the two halves of the Lysep column were separated. The
bottom half containing the liquid solution was kept and the top half containing what was left of
the meat sample was discarded. The bottom tube was placed into row S of the Holder for the
AutoMate Express instrument. An empty microfuge tube was placed into row E of the Holder
and a clean tip and tip holder was placed into row T2. A second tray held the PrepFiler Express
cartridges placed into it, matching each cartridge to a sample tube. A negative sample control
was also loaded into the extraction run. Both of the trays were then loaded into the AutoMate
Express instrument. The program card was then placed into the instrument as well and the
AutoMate Express bodily fluid protocol was selected and carried out. This program took about
30 minutes to complete. The extraction system of the AutoMate Express is a magnetic bead
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separation technique. After the extraction run had been completed the extracted DNA of each
of the samples was now in the 1.5ml microfuge tubes that were loaded into the tray before the
run began. Each of the tubes was then labelled according to the sample that it contained
before. The tubes were labelled PS 1, 2, 3, 4 and 5. PS was the identifier for the meat samples
that went through the bodily fluid extraction procedure. All of the samples were then placed
into the freezer to be stored at -18oC until the samples were needed for quantitation.
The blood samples were also extracted using the bodily fluid extraction methods as
outlined above. 40µl of blood was placed into each Lysep column. Two microfuge tubes were
created for each pig blood sample. This created 10 samples that were to be extracted. The
tubes were labelled PBL (pig blood) 1.1, 1.2, 2.1…. 5.2. two mixtures were also made with the
pig’s blood. Using pig blood sample 1 and 3 two mixtures were created with equal quantities of
each of the blood samples (20µl of each sample). These two samples were labelled PBLM (Pig
Blood Mixture) 1.1 and 1.2.
The dilution series for the Blood was created by having a set of blood mixtures that
were 1in1, 1in10, 1in25, 1in50, 1in100 and 1in200. The dilution series was done with the blood
samples labelled 943:01, (pig 1) 945:02, (pig 2) and 946:03 (pig 3). The 1in1 mixture was made
by extracting 100µl of blood into a microfuge tube. The 1in10 dilution was made by adding 10µl
of pig blood into 90µl of water. The 1in25µl dilution was created by adding 5µl of pig blood in
120µl of water. The 1in50 dilution was created by adding 5µl of pig blood in 245µl of water. The
1in100µl dilution was created using 5µl of pig blood and 495µl of water. The 1in200 dilution
was created using 5µl of pig blood and 995µl of water. This created 18 samples, six samples for
each of the three different pig blood samples. After the dilutions were created the microfuge
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tubes containing the diluted blood samples were vortexed in the mini vortexer (Fisher
Scientific, Waltham, Massachusetts, United States) and spun down in the mini centrifuge (E&K
Scientific, California United States). After the centrifugation 75ml of each of the samples was
aliquoted by dotting them onto Whatman FTA paper (Whatman, Maidstone, United Kingdom).
The dotting created circles approximately 2.5cm in diameter. The samples were left to dry for
about an hour in the DNA extraction room at room temperature. After the hour was up, two
punches were taken of each of the samples for extraction. The punch was circular and was
0.5cm in diameter. The puncher was cleaned with a water and bleach solution between each
punch. The two punches were placed in a Lysep column and the extraction procedure that was
outlined above for the bodily fluid DNA extraction technique was carried out.
The remaining five meat samples that were teased off of the pork bone went through
the BTA extraction procedure. This procedure was very similar to the bodily fluid extraction
procedure, however, instead of the sample being placed into the Lysep column the thawed
meat samples were placed into the provided bone and tooth lysate tubes. Next, 230µl of
PrepFiler BTA lysis solution was added to the tubes with the sample. The BTA lysis solution
consisted of 220µl of PrepFiler BTA lysis buffer, 3µl of DTT and 7µl of Proteinase K. The
thermomixer was then heated to 56oC and the samples were placed into the Eppendorf
thermomixer for 18 hours at 750rpm. After 18 hours of uncubation, the lysate tubes were
removed from the Thermomixer, they were centrifuged in the Eppendorf Centrifuge 5424
(Eppendorf, Hamburg Germany) for 2 minutes at 10,000x the force of gravity. These samples
were labelled BTA 1 through 5.
The Bone samples were prepared with the BTA extraction as well. The small fragments
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of bone dust weighing approximately 0.6g were placed into the bone and tooth lysate tubes.
The procedure as outlined above was carried out for the extraction of the DNA from the Bone
samples. The extracted DNA from the bone samples was labelled BS (Bones Sample) 1, 2, 3, 4,
and 5.
A complete list of all of the samples that had their DNA extracted can be found in
Table 2.1.
2.3 Quantification of DNA
After the extraction of DNA was completed, a quantitation of the amount of DNA in
each of the extracted samples was performed. The first puantitation was done using the ABI
7500 Real-Time PCR instrument (Applied Biosystems, Foster City, California) and the
Quantifiler® Human DNA Quantification Kit from Thermo Fisher Scientific [15], which uses a
human-specific assay that targets the 18S region of the chromosome. The quantitation was
done to demonstrate that the pig DNA would not be recognized by the human specific primers
in the Assay mix. The components of the reaction mix that were added to the sample in real
time PCR instrument are outline in Table 2.2.
The quantification for the amount of pig DNA in each of the extracted samples was done
with the NanoDrop 8000 spectrophotometer (Thermo Scientific Waltham, Massachusetts,
United States)[16]. A 2µl volume of each of the samples was pipetted onto one of the eight
optical surfaces of the device. The device then measures the absorbance of each of the samples
in the 260-280nm wavelength. The instrument automatically generates a curve and calculates
the concentration of the DNA within each of the samples in ng per µl.
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Using the results from the NanoDrop 8000 the samples were diluted to a concentration
of approximately 2.5ng/ml. The list of the dilutions are summarized in Table 2.3.
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Table 2.1. All samples subjected to DNA extraction.
Muscle samples Bone samples Blood samples
PS 1 BS 1 PBL 1.1 ,1.2
PS 2 BS 2 PBL 2.1, 2.2
PS 3 BS 3 PBL 3.1, 3.2
PS 4 BS 4 PBL 4.1, 4.2
PS 5 BS 5 PBL 5.1, 5.2
PS Blank BS Blank Pig 1 dilution series
BTA 1 Pig 2 Dilution series
BTA 2 Pig 3 Dilution series
BTA 3 PBLM 1.1, 1.2
BTA 4 PBL Blank
BTA 5
BTA Blank
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Table 2.2. Reagents and volumes for one sample amplified on the Applied Biosystems real time
PCR instrument.
Reagent Volume (µl)
Quantifiler® Human Primer Mix 10.5
Quantifiler® PCR Reaction Mix 12.5
Sample 2.0
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Table 2.3. Dilutions of the extracted samples to make the approximate concentration of DNA
2.5ng per µl.
Sample Sample volume (µl) TE buffer volume (µl)
PS 1 2.2 122.8
PS 2 2.3 122.7
PS 5 2.1 47.9
BTA 1 4.3 120.7
BTA 3 2.1 47.9
BTA 5 2.6 47.4
BS 1 2.0 248.0
BS 3 2.1 47.9
BS 5 5.0 1245.0
PBL 1.1 2.5 47.5
PBL 2.1 4.9 20.1
PBL 3.1 3.0 22.0
PBL 4.1 5.5 19.5
PBL 5.1 2.8 22.2
PBLM 1.1 2.1 22.9
PBLM 1.2 2.6 22.4
Pig 1: 1:1 6.1 18.9
Pig 1: 1:10, 1:25, 1:50, 1:100, 1:200
2.5 0
Pig 2: 1:1 3.5 21.5
Pig 2: 1:10, 1:25, 1:50, 1:100, 1:200
2.5 0
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2.4 PCR Amplification and Electrophoresis
The next step in the process was to amplify the diluted sample using PCR (the
polymerase chain reaction). The Kit that was purchased from Germany was called the Animal-
type pig kit. The instructions in the user manual [13] called for the reaction volume to be 25µl.
Due to limited resources it was decided to make the reaction volume 12.5µl in order to be able
to amplify twice as many samples. The volumes of each of the reagents that were used for one
PCR reaction were: 6.05µl of nuclease free water, 2.5µl primer mix D, 1.25µl of primer mix,
0.2µl of Taq 2 DNA polymerase and 2.5µl of sample to be amplified.
Once prepared, the samples were placed into the Applied Biosystems 9700 Thermal
cycler (Applied Biosystems, Foster City, California, United States). The instrument had been
programmed to follow the heating and cooling cycles per the Animal-type kit [14]. One
complete heating and cooling cycle is as follows: start at 94oC and hold for 4 minutes (hot start),
then 94oC for 20 seconds, then 60oC for 40 seconds followed by 72oC for 30 seconds, this was
repeated for 30 cycles. Then the third portion of the cycle was 70oC for 60minutes and then a
decrease to 10oC to be held until the samples were removed from the thermal cycler.
In order for the samples to be run on the 3130 Genetic Analyzer (Applied Biosystems,
Foster City, California, United States) a spectral calibration had to be performed. The spectral
calibration was done to make sure that the dyes were not overlapping in the system. The four
dyes that are used in the Biotype, Animal-type Pig kit are 6-FAM, HEX, NED, and ROX [17]. The
spectral calibration standard [SD-30 Matrix Standard Kit (Dye set D) Applied BiosystemsTM] was
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made with 47.5µl of Hi-Di formamide (Manufacturer) and 2.5µl of matrix standard. 10µl of the
master mix was aliquoted into 4 adjacent wells in a 96 well plate.
After the spectral calibration had been completed the samples were prepared to go into
the genetic analyzer. The samples were added to a 96 well plate. Before the addition of the
samples the wells that were to house a sample, had 12µl of master mix added to them. The
master mix was created with 12.3µl of Hi-Di formamide and 0.2µl of size standard 550 (ROX,
provided in the Biotype Animal-type Pig Kit) per sample that would be injected. Added to the
samples were four allelic ladders, two Negative amplification blanks and one positive
amplification control. In total 32 samples were run.
Once the raw data came from the 3130 Genetic Analyzer it was transferred to another
computer via USB. The other computer had GeneMapper® ID-X v1.4 software from
ThermoFisher Scientific (Thermo Scientific Waltham, Massachusetts, United States). The panels
and bins that were required to analyze the raw data from the genetic analyzer were
downloaded from the Animal-type website [14] and modified for compatibility with the
genotyping software version. The panels and bins were then imported into the GeneMapper ID-
X software.
The STR profiles were then analyzed and cleaned-up by means of the analyst clicking off
peaks that were noise, stutter, pull-up or other kinds of artifacts that were found within the
profiles.
Using the cleaned-up data the two most dissimilar profiles were chosen to create the
mixture profiles. The two profiles that were the most dissimilar were the sample from the
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muscle tissue/bone tissue and pig 946:03 (pig blood 3). Using the extracted DNA from the two
previously named diluted samples a mixture profile was created by amplifying mixtures of DNA
from the two sources in varying proportions. The mixture profile entailed creating a mixture of
different ratios. The ratios that were chosen were: 5% pig blood 3 to 95% bone sample, 10% pig
blood, 25% pig blood, 50% pig blood, 75%pig blood, 90% pig blood and 95% pig blood. In total
there were seven mixture profiles developed.
The extracted diluted samples then went through the same processes listed above for
PCR amplification and capillary electrophoresis. The raw data was then brought over to the
gene mapper IDX software to have the profiles examined and cleaned up for examination.
2.5 Statistical Tests Used
The statists were calculated partially by hand and also using an excel workbook to
calculate the complex and larger numbers.
To determine whether or not there was a statistical difference between the BTA
extraction and the bodily fluid extraction a Wilcoxon Test for nonparametric data was
performed.
The mean height of the heterozygous and homozygous alleles was calculated for each of
the profiles of the dilution series. A Kruskal Wallis test was used to see if there was a significant
difference between the mean peak heights of the alleles for the different dilution
20
concentrations in the series. To determine where any statistical difference may lay a Nemenyi
test was performed.
The mean stutter peak height ratio was calculated for many different profiles and many
different alleles. Once the mean was calculated the standard deviation was calculated. The
average stutter peak height plus 3 standard deviations was set as the stutter peak height cut
off.
21
CHAPTER 3
RESULTS
3.1 Results
The first set of results that were obtained was from the quantification of the pig DNA
with the human primers using the QuantiFiler Human assay in the ABI 7500 real time PCR
instrument. All of the quantifications came back with a value of zero.
The second quantitation that took place was done with the NanoDrop 8000. In this
quantitation the muscle and bone samples were run to determine the quantity of DNA in the
extracted samples. These quantitation data are listed in Table 3.1. Some samples were run
twice. The Wilcoxin test found that there was a significant difference at the 0.025 confidence
level between the concentrations of the BTA extraction when compared to the bodily fluid
extraction.
The third quantitation was with the blood samples; also using the NanoDrop 8000. In
this quantitation there were two mixtures, the normal blood samples and the dilution series.
These results can be found in Table 3.2.
A positive control that came with the kit was run (Figure 3.1) As well as a negative
amplification blank (Figure 3.2). No alleles were found in the Negative amplification blank.
One of the replicate STR profiles generated from the meat sample and from the Bone
sample are shown in (Figures 3.3 and 3.4 are respectively).
22
The Profiles of the dilution series (Figures 3.5-3.10) are present to demonstrate the
effects the dilution had on the RFU (Relative Fluorescence unit) heights. As the sample became
more diluted the RFUs decreased for each of the peaks. Additionally, as the sample became
more diluted allele drop out started to occur.
The average peak heights of the heterozygous and the homozygous alleles are shown
for each of the dilutions in the dilution series (Table 3.3) to compare the affect dilution had on
the DNA profile. The Kruskal Wallis test found here to be a significant difference at the 0.025
confidence level. The Nemenyi test found that there was a significant difference between the
two most dilute samples (1:100 and 1:200) and the least diluted samples (1:1 and 1:10).
23
Table 3.1. Concentration of DNA in each of the muscle and bone samples.
Sample Concentration of DNA (ng/µl)
1st round 2nd round
PS 1 8.445 57.59
PS 2 17.68 55.46
PS 3 36.27 80.70
PS 4 2.981 32.82
PS 5 13.26 23.90
PS Blank -2.743 -2.660
BTA 1 30.46 29.13
BTA 2 23.51 11.46
BTA 3 22.91 24.38
BTA 4 6.559 10.72
BTA 5 57.16 19.04
BTA B -1.116 -2.340
BS 1 139.2 128.2
BS 2 256.2 247.5
BS 3 27.62 24.08
BS 4 429.6 n/a
BS 5 415.8 n/a
BS B -3.777 n/a
24
Table 3.2. Concentration of the extracted DNA from the pig blood samples
Sample Concentration of DNA (ng/µl)
PBL 1.1 19.61
PBL 2.1 5.065
PBL 3.1 8.272
PBL 4.1 4.550
PBL 5.1 9.087
PBLM 1.1 11.76
PBLM 1.2 9.652
Pig 1 1:1 2.223
Pig 1 1:10 7.643
Pig 1 1:25 24.76
Pig 1 1:50 32.38
Pig 1 1:100 5.285
Pig 1 1:200 27.43
Pig 2 1:1 0.5572
Pig 2 1:10 24.88
Pig2 1:25 10.00
Pig 2 1:50 11.21
Pig 2 1:100 38.95
Pig 2 1:200 7.661
25
Figure 3.1. STR profile of the positive control that came in the Animaltype pig kit. This sample
was electro-kinetically injected for 5 seconds.
26
Figure 3.2. The Negative amplification blank STR profile. This sample was electro-kinetically
injected for 5 seconds.
27
Figure 3.3. STR profile for the meat sample that was obtained from the pork bone using the BTA
extraction. This sample was electro-kinetically injected for 5 seconds.
28
Figure 3.4. STR profile of a bone sample that was taken from the pork bone. This sample was
electro-kinetically injected for 5 seconds.
29
Figure 3.5. STR profile of pig blood sample 1 with no dilution. This Sample was electro-
kinetically injected for 5 seconds.
30
Figure 3.6. STR profile of pig blood sample 1 with a 1:10 dilution. This sample was electro-
kinetically injected for 5 seconds.
31
Figure 3.7. STR profile of pig blood sample 1 with a 1:25 dilution. This sample was electro-
kinetically injected for 5 seconds.
32
Figure 3.8. STR profile of pig blood sample 1 with a 1 in 50 dilution. This sample was electro-
kinetically injected for 5 seconds.
33
Figure 3.9. STR profile of pig Blood sample 1 with a 1 in 100 dilution. The sample in this profile
was electro-kinetically injected for 10 seconds.
34
Figure 3.10. STR profile of pig blood sample 1 with a 1:200 dilution. This sample was electro-
kinetically injected for 10 seconds.
35
Table 3.3. Average heterozygous and homozygous Peak heights for each of the dilutions.
Dilution Mean Homozygotic peak height (RFU)
Mean heterozygotic peak height (RFU)
1:1 4349 2510
1:10 1600 1032
1:25 1309 828
1:50 1248 890
1:100 298 318.4
1:200 259.4 180
36
The mixture series ranging 5% pig blood to 50% pig blood are shown (Figures 3.11-3.14)
to allow a visual representation of when a mixture can still be detected in a DNA profile. The
mixture is between two different pig’s DNA allowing up to four alleles to be seen at some of the
loci.
To determine the cut off for stutter height especially for a mixture profile, the mean
stutter height ratio was determined for each locus (Table 3.4).
37
Figure 3.11. 50%/50% mixture of DNA between pig blood sample 3 and bone sample 1
38
Figure 3.12. 25%/75% mixture of DNA between pig blood sample 3 and bone sample 1
39
Figure 3.13. 10%/90% mixture of DNA between Pig blood sample 3 and bone sample 1
40
Figure 3.14. 5%/95% mixture of DNA between pig blood sample 3 and bone sample 1
41
Table 3.4. Stutter to Peak height ratio of the different loci in the STR profiles.
Locus Mean Stutter Ratio % Standard Deviation of stutter ratio
SBH2 25.61 4.74
SBH18 11.89 2.39
SBH4 11.73 3.07
S0665 5.69 1.04
SBH20 20.48 9.95
SBH1 7.7 2.13
SBH10 20.97 5.41
SBH13 11.49 2.96
387A12F 6.97 3.22
SBH22 8.4 1.03
SBH 19 9.29 2.38
42
CHAPTER 4
DISCUSSION
4.1 Points of discussion
The main goal of this study was to determine whether or not an STR profile could be
created from pig DNA using the equipment that was commonly found in a forensic DNA
laboratory; more specifically using equipment found in the Laurentian University Forensic DNA
Laboratory. This goal was accomplished as 6 individual STR profiles were developed from the Kit
plus the positive control that came with the kit. The positive control that came with the kit also
came with a reference of the STR profile of the Positive control sample. The profile that was
generated (Figure 3.1) was identical to the profile that the company gave [19]. Every allele at all
of the loci were the same. This indicated that the equipment was functioning properly as the
profile that was developed was the same as profile that was supposed to be generated.
Additionally, all of the samples that were absent of DNA did not yield a profile (Figure 3.2).
Upon extracting DNA from the pork bone the samples, quantitative PCR was run in the
on the samples using the primers that were designed for humans in the QuantiFiler Human kit.
Upon the completion of the quantitative PCR there was found to be no detected human DNA in
any of the samples. Yet, when the samples were run on the NanoDrop 8000 all of the samples
contained DNA. Furthermore, an STR profile was developed for all the samples. The reason that
the real time PCR instrument did not recognize that there was DNA in the sample was because
the primers did not bind to the pig DNA. Because the primers did not bind to the pig DNA it
showed that they were in fact human-specific. This was good news as part of the reason this
43
research is being conducted is because a laboratory may wish to conduct research and profile
case samples. Normally the analyst would have to worry about cross-contamination between
the research samples and the case samples. However, if the researcher was using pig DNA the
primers would not recognize the pig DNA. In addition, if there happened to be any cross-
contamination, the pig DNA would not affect the result as the human specific primers would
not be able to bind to the pig DNA.
During this research, one of the questions that was raised was whether or not a muscle
tissue sample would be best extracted using the PrepFiler bodily fluid extraction procedure or
the PrepFiler BTA extraction procedure. In order to investigate this question, five meat samples
from the same piece of meat were extracted with each extraction technique. Once the DNA had
been extracted the samples went through quantification with the NanoDrop 8000. The mean
concentration of DNA in the 5 samples that went through the bodily fluid extraction procedure
was 50.1ng/µl, whereas; the mean concentration of the five samples that went through the BTA
extraction procedure was 28.1ng/µl. The Wilcoxon test showed that there was a significant
difference to the 0.025 confidence level.
Upon statistical evaluation it was found that the two extraction procedures were
significantly different from one another in the quantity of DNA that was produced from the
extraction. It was expected that the BTA extraction technique would yield more DNA because it
was designed for more complex tissue samples and had the addition of Proteinase K that aids in
the breaking down of proteins. Even though the addition of the Proteinase K and the longer
time in the thermomixer the BTA extraction was less efficient.
44
When observing the samples after they were removed from the thermomixer for each
of the extraction procedures, the BTA extraction had degraded the sample of meat more than
the bodily fluid extraction had. For this reason it was hypothesized that the BTA would yield
more DNA than the bodily fluid extraction procedure. This was not the case. The bodily fluid
extraction appeared to be more efficient in releasing the DNA from solution.
One possible reason for the BTA extracting less DNA than the bodily fluid extraction is
that the BTA actually destroyed the DNA that was in the sample. It is possible that the muscle
tissue was not rigid enough to protect the DNA that the procedure actually extracted and
denatured/destroyed the DNA when lysing the cells. Then when the sample solution went
through the extraction in the AutoMate Express only a portion of the DNA that was lysed from
the cells was able to bind to the magnetic particle suspension and the rest was washed out with
the buffer and contaminants.
The dilution series was conducted to examine the ability of the method to analyze DNA
in low quantities and also to determine at what concentration alleles started to drop out of the
profile. Alleles first started to drop out of the profile at a concentration of approximately 1:100.
Even when the electro-kinetic injection was 20 seconds long, the longest recommended from
the kit, there were still alleles missing. One allele that dropped out was allele 37 at locus SBH20
(Figure 3.9). The statistical analysis, Kruskal Wallis and Nemenyi tests, were done to see which
dilutions were significantly different. The 1:1 and 1:10 ratio were significantly different from the
1:100 and the 1:200 profiles, respectively. The 1:1 and 1:10 ratios had much larger peak
heights than the 1:100 and 1:200 profiles. This makes sense as the alleles were beginning to
45
drop out at the concentration of 1:100 and the injection times had to be increased from 5
seconds to 10 and 20 seconds to develop any kind of profile.
The mixture analysis was done to see at what relative proportion the minor component
of the DNA profiles could be detected. At a 10% pig blood to 90% bone sample and 5% blood
sample to 95% bone sample a couple of alleles at some loci are visible but not a whole second
profile (Figures 3.13 and 3.14). From these results it was determined that the presence of a
mixture could be determined all the way down to a 1 in 20 mixture component but a reliable
profile could not be generated. A complete second profile was visible at a 25% pig blood to 75%
bone sample concentration though.
The mean stutter height to peak height ratio was calculated. The mean stutter peak
height ratio was found to be 12.75% with a standard deviation of 3.48. The stutter peak height
ratio was very close to the manufactures recommendation of 13% for the kit. Three standard
deviations were added to the mean stutter peak height ratio. The result of the 12.75% plus 3
standard deviations is a value of 23.2%. With this it was concluded that any stutter peak that
was greater than 23.2 % of the actual peak could indicate that there was the possibility of
another source of DNA adding to the stutter peak. This information provides a guide line for
mixture analysis. It would allow an analyst to have a specific cut-off of what should be
considered another peak from a second possible source.
The guidelines that were being followed for this research were the guidelines of a
validation study that were set out by the Scientific Working Group on DNA Analysis Methods
(SWGDAM) [11]. The necessary components of a validation are: peer reviewed articles,
46
sensitivity studies, species specificity, precision and accuracy and case type samples. A current
article outlining the ability of the AnimalType kit is Caratti et al. [5], it shows the ability of the kit
to differentiate pigs and the power of exclusion and discrimination of the kit. The species
specificity was not shown in the current study. In order to accomplish this, human samples
would have to be run using the kit and determine if a profile could be developed from the
human samples using the pig primers for amplification. The sensitivity studies were shown in
the dilution series as well as the mixture analysis. Extremely dilute samples were run and a
profile could still be generated at a dilution of 1 in 200. Additionally, the presence of a mixture
could be detected even at 1 in 20 concentration of one type of sample in another. The accuracy
and precision of the kit and the procedure was demonstrated through the positive control and
the duplicates of the muscle, bone and blood samples that were run. The positive control was
identical to the manufacturer’s reference profile. This demonstrated accuracy. Precision was
demonstrated when the duplicates of the bone and meat samples all generated the same
profile and because the bone and meat samples had the same profile of each other because
they both came from the same source of DNA. The case type samples requirement of the
SWGDAM guidelines was half fulfilled through the mixture and dilution series that were run.
The dilution and mixture series showed that the kit and procedure can handle extremely dilute
samples as well as mixtures which are common in forensic case work. Mixtures are common in
sexual assault type cases and dilutions are common when an individual tries to clean up after
they have committed a crime. The case type sample requirement is only half fulfilled because
degraded samples and samples on different substrates are also part of case type samples and
these types of samples were not tested in this study.
47
4.2 Limitations of the Study
This study was limited in the number of samples that were run as well as the ability of
the study to do an accurate quantification. In order for the study to be strengthened many
more samples will need to be run. Some of the samples that will need to be tested are
degraded DNA samples, more blanks to determine the analytical and stochastic thresholds as
well as samples on different substrates. These additional samples will advance the validation to
show that case type samples can be analyzed as well as determine the background noise levels
and to determine at which RFU level a peak in the STR profile should be considered an allele.
Additionally, The stochastic threshold should be decided on. The kit recommends a value of 75
RFU but on one of the dilutions there was a peak greater than 75 RFU and the other alle that
was supposed to be present had dropped out. This means that a stochastic threshold greater
than 75 RFU will likely be needed. Human samples will also need to be run to show that the
primers in the kit do not bind with human DNA and create a profile. Pig samples should also be
amplified with the current human primers. This would show that the pig DNA would not
generate a profile with the human specific primers used in the lab.
The quantification in this research was very limited because the NanoDrop 8000 was not
very precise. The values varied from run to run even from the same sample by more than
10ng/µl. An accurate quantification will need to be used so that the dilutions for the
amplification for capillary electrophoresis are accurate. In this study due to the fact the
concentration of DNA was only an estimation the dilutions were not very accurate and many of
48
the samples appeared to be overloaded, having an excessive amount of DNA and leading to
numerous artifacts in the profiles.
49
CHAPTER 5
CONCLUSIONS
5.1 Summation
This study completed the primary task that it set out to achieve. The task that was
achieved was the development of an STR profile using pig DNA with the same methods and
procedures that are currently in practise for forensic case work. The study also succeeded in
completing most of the necessary requirements for the procedure to be validated and put into
practise in a standard DNA laboratory. The parts of the validation that the research completed
was that a stutter peak larger than 23.2% of the actual peak should be considered a possible
source of another DNA contributor, a partial profile can be determined even if the DNA is
extremely diluted and it is possible to determine if a mixture is present even if the minor
contributor of the DNA mixture is only 5% of the overall mixture. This research project
demonstrated the viability of using pig DNA as a research tool in a forensic laboratory. The pig
DNA would not cross contaminate any active case work that may be taking place. Additionally,
many other forensic disciplines are currently using pigs as research tools and the use of pig DNA
would allow colleagues to collaborate and work together in research.
5.2 Recommendations
The next step for research in this area would be to develop a primer that binds to
double stranded pig DNA. This primer would need to have a sequence of DNA that is present in
50
all pigs and that is specific to pigs. The primer would need to be present in all pigs and be
specific to pigs because it would be used for the real time PCR instrument. The development of
a fluorescent primer that can bind to a certain region of the nuclear genome the pig would
allow for accurate quantification of the pig DNA. Accurate quantification is an essential part of
the typical flow of samples in a forensic laboratory and thus, would be needed for the
procedure to be validated.
Another step that should be accomplished would be the optimization of the PCR
amplification before being run in electrophoresis. The PCR amplification needs to be optimized
because there appeared to be a lot of slippage that occurred during the PCR. This was shown
when the mean stutter height ratio was 25% (Table 3.5) at the locus SBH2. The stutter peaks
are too high and can almost be misinterpreted as being a mixture with peaks from a minor
contributor.
51
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